Differential tagging with isotopic reagents, such as isotope-coded affinity tags (ICAT) (1) or the more recent variation that uses isobaric tagging reagents, iTRAQ (Applied Biosystems, Foster City, Calif.), followed by multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis is a powerful methodology in the search of biomarkers for various disease states.
Endometrial carcinoma (EmCa), a cancer of the lining of the uterus, is the fourth most common cancer in Canadian women (4). Current methods of diagnosis rely on invasive techniques—biopsy and curettage—and no screening is available. A panel of biomarkers that helps in early diagnosis would, therefore, be useful, especially for highrisks groups, e.g., women who are on Tamoxifen treatment or have hereditary nonpolyposis colorectal cancer syndrome. Although the eventual diagnostic testing for such biomarkers would be most facile from bodily fluids, such as blood or urine, the iTRAQ experiments performed thus far have been on resected EmCa from uterine tissues (hysterectomy specimens) (2, 3). The rationale for this approach is that the concentration of any biomarker is most likely highest in the cancerous tissue itself, and not when diluted in the bodily fluids, thus facilitating discovery. In addition, the use of the cancerous tissue reduces the intrinsic need to demonstrate that any differentially expressed protein detected does indeed originate from the endometrial cancer. By contrast, the origins of differentially expressed protein in the blood could include a variety of potential sites other than the actual tumor. The use of homogenized tissues yields a heterogeneous sample with the proteome being contributed by the stroma, vasculature, blood, and malignant/benign epithelium. This heterogeneity may attenuate, and even mask, the variation in protein expression levels characteristic of cancerous epithelial cells. One remedy for this drawback is the use of laser capture microdissection (LCM) to procure the specific, malignant epithelial cells from the samples (5). This approach, however, is not practical, when 103-104 cells per sample are required for current proteomic techniques, in a global biomarker discovery strategy. Thus far, the types of differentially expressed proteins discovered (2, 3) are primarily medium- to high-abundance proteins, as universal detection methods, including the MS/MS technologies that were employed, are much more efficient in detecting major rather than minor components in a complex mixture.
A strategy in the search of EmCa markers requires a comparison between the cancerous endometrium and the two major phases, proliferative and secretory, of the normal reproductive-aged endometrium (3, 6). The multiplexing ability afforded by the iTRAQ reagents, which are available in four different tags or flavors, is well suited for such a simultaneous comparison, especially in view of the fact that endometrial carcinoma itself can have two distinct morphologic and physiologic types. Type I cancers are endometrioid in histologic typing, well-differentiated, and estrogen-dependent; and have typically a better prognosis. By contrast, Type II carcinomas are serous and clear cell carcinomas, hormone-independent, and aggressive; and have generally a poorer clinical outcome (7).